Minocycline prevents hypoxia-induced seizures

Abstract

Severe hypoxia induces seizures, which reduces ventilation and worsens the ictal state. It is a health threat to patients, particularly those with underlying hypoxic respiratory pathologies, which may be conducive to a sudden unexpected death in epilepsy (SUDEP). Recent studies provide evidence that brain microglia are involved with both respiratory and ictal processes. Here, we investigated the hypothesis that microglia could interact with hypoxia-induced seizures. To this end, we recorded electroencephalogram (EEG) and acute ventilatory responses to hypoxia (5% O₂ in N₂) in conscious, spontaneously breathing adult mice. We compared control vehicle pre-treated animals with those pre-treated with minocycline, an inhibitory modulator of microglial activation. First, we histologically confirmed that hypoxia activates microglia and that pre-treatment with minocycline blocks hypoxia-induced microglial activation. Then, we analyzed the effects of minocycline pre-treatment on ventilatory responses to hypoxia by plethysmography. Minocycline alone failed to affect respiratory variables in room air or the initial respiratory augmentation in hypoxia. The comparative results showed that hypoxia caused seizures, which were accompanied by the late phase ventilatory suppression in all but one minocycline pre-treated mouse. Compared to the vehicle pre-treated, the minocycline pre-treated mice showed a delayed occurrence of seizures. Further, minocycline pre-treated mice tended to resist post-ictal respiratory arrest. These results suggest that microglia are conducive to seizure activity in severe hypoxia. Thus, inhibition of microglial activation may help suppress or prevent hypoxia-induced ictal episodes.

Keywords: SUDEP; hypoxia; microglia; minocycline; seizure.

Figure 1

Morphology of Iba-1 positive cells in the piriform cortex of mice in four conditions: with and without minocycline, without and with hypoxic (7% O₂) loading. (A) Room air without minocycline: ramified non-active cells. (B) Room air with minocycline: ramified cells. (C) Hypoxia without minocycline: activated ovoid cells. (D) Hypoxia with minocycline: mostly ramified Iba-1 positive cells intermingled with some ovoid cells. Minocycline suppressed hypoxia-induced microglial activation to some extent in the epileptogenic piriform cortex. The panels’ left-sided rectangles were blown-up in the corresponding right panels. The scale bars are all 200 μm.

Figure 2

Representative raw recordings of respiratory flow (inspiration upward) and EEG signal in mice without and with minocycline in room air and 5% hypoxia showing the initial ventilatory augmentation and late ventilatory fall-off. Minocycline failed to influence the hypoxic ventilatory response.

Figure 3

Time courses of respiratory variables in the vehicle- and minocycline pre-treated conditions before and during 5% hypoxia. (A) Minute ventilation (⩒_E). (B) Tidal volume (V_T) in each mouse. (C) Respiratory rate (RR). As compared to vehicle-pre-treated mice, minocycline-pre-treated mice tended to resist respiratory depression and respiratory arrest during the late hypoxic phase. MINO, minocycline.

Figure 4

Inhibition of microglial activation delayed the occurrence of seizures. (A) Representative raw recordings of respiratory flow (inspiration upward) and EEG signals in hypoxia experiments without and with minocycline. Seizures were accompanied by high amplitude aberrant waves in EEG (the onset of seizures was indicated by downward arrows). The time of respiratory arrest was indicated by an asterisk. (B) Time from the onset of hypoxia to seizures in vehicle and minocycline pre-treated mice. Time to seizure was significantly longer in the latter group of mice (Mann-Whitney U test). MINO, minocycline. ^*p < 0.05.

Figure 5

Kaplan−Meier curves showing the time from hypoxia onset to respiratory arrest in the vehicle (saline) and minocycline pre-treated mice; 10 mice each. There were no significant differences between the two groups. MINO, minocycline.